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High CD206 levels in Hodgkin lymphoma-educated
macrophages are linked to matrix-remodeling and
lymphoma dissemination
Annekatrin Arlt1,2, Frederike von Bonin1, Thorsten Rehberg3, Paula Perez-Rubio2,3,
Julia C. Engelmann2,3*, Katharina Limm4 , Sarah Reinke5, Christian Dullin6, Xueni Sun4,
Rieke Specht1, Markus Maulhardt1, Franziska Linke1†, Gertrude Bunt7, Wolfram Klapper5,
Martina Vockerodt8, Jo € rg Wilting2,8, Tobias Pukrop2,9, Katja Dettmer2,4, Wolfram Gronwald2,4,
Peter J. Oefner , Rainer Spang2,3 and Dieter Kube1,2
4

1 Clinic of Hematology and Medical Oncology, University Medical Centre Go €ttingen, Germany
2 Network BMBF eMed MMML-Demonstrators, Regensburg, Germany
3 Statistical Bioinformatics, Institute of Functional Genomics, University of Regensburg, Germany
4 Institute of Functional Genomics, University of Regensburg, Germany
5 Department of Pathology, Hematopathology Section, UKSH Campus Kiel, Germany
6 Institute of Diagnostic and Interventional Radiology, University Medical Centre Go €ttingen, Germany
7 Clinical Optical Microscopy, Institute of Neuropathology, University Medical Centre Go €ttingen, Germany
8 Institute of Anatomy and Cell Biology, University Medical Centre Go €ttingen, Germany
9 Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Germany

Keywords Macrophages (Mφ) are abundantly present in the tumor microenvironment

CD206; lymphoma; macrophages; tumor
microenvironment
Correspondence
D. Kube, University Medical Centre of the
Georg-August University Go€ttingen, Clinic of
Hematology and Medical Oncology, Robert-
€ttingen, Germany
Koch-Str. 40, 37075 Go
Tel/Fax: +49 551 395307
E-mail: dieter.kube@med.uni-goettingen.de
Present address
*NIOZ Royal Netherlands Institute for Sea
Research, Utrecht University, Utrecht, The
Netherlands
†
School of Medicine, Queen’s Medical
Centre Campus, University of Nottingham,
UK
(Received 5 August 2019, revised 8
November 2019, accepted 9 December
2019)
doi:10.1002/1878-0261.12616
and may predict outcome in solid tumors and defined lymphoma subtypes.
Mφ heterogeneity, the mechanisms of their recruitment, and their differen-
tiation into lymphoma-promoting, alternatively activated M2-like pheno-
types are still not fully understood. Therefore, further functional studies
are required to understand biological mechanisms associated with human
tumor-associated Mφ (TAM). Here, we show that the global mRNA
expression and protein abundance of human Mφ differentiated in Hodgkin
lymphoma (HL)-conditioned medium (CM) differ from those of Mφ edu-
cated by conditioned media from diffuse large B-cell lymphoma (DLBCL)
cells or, classically, by macrophage colony-stimulating factor (M-CSF).
Conditioned media from HL cells support TAM differentiation through
upregulation of surface antigens such as CD40, CD163, CD206, and PD-
L1. In particular, RNA and cell surface protein expression of mannose
receptor 1 (MRC1)/CD206 significantly exceed the levels induced by classi-
cal M-CSF stimulation in M2-like Mφ; this is regulated by interleukin 13
to a large extent. Functionally, high CD206 enhances mannose-dependent
endocytosis and uptake of type I collagen. Together with high matrix met-
alloprotease9 secretion, HL-TAMs appear to be active modulators of the
tumor matrix. Preclinical in ovo models show that co-cultures of HL cells
with monocytes or Mφ support dissemination of lymphoma cells via lym-
phatic vessels, while tumor size and vessel destruction are decreased in

Abbreviations
APC, allophycocyanin; CAM, chick chorioallantoic membrane; CM, conditioned medium; DDA, data-dependent acquisitions mode; DLBCL,
diffuse large B-cell lymphoma; DSMZ, Deutsche Sammlung fu €r Mikroorganismen und Zelllinien; FACS, fluorescence-activated cell sorting;
FFPE, formalin-fixed paraffin-embedded; HL, Hodgkin lymphoma; HRP, horseradish peroxidase; M-CSF, macrophage colony-stimulating
factor; MMP, matrix metalloprotease; MRC1, mannose receptor 1; Mφ, macrophages; PBMC, peripheral blood mononuclear cells; PE,
phycoerythrin; PTA, phosphotungstic acid; RPMI, Roswell Park Memorial Institute; TAM, tumor-associated Mφ; TMA, tissue microarray;
TME, tumor microenvironment.

Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 571
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.
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Hodgkin lymphoma-educated macrophages and CD206 A. Arlt et al.

comparison with lymphoma-only tumors. Immunohistology of human HL

tissues reveals a fraction of cases feature large numbers of CD206-positive
cells, with high MRC1 expression being characteristic of HL-stage IV. In
summary, the lymphoma-TAM interaction contributes to matrix-remodel-
ing and lymphoma cell dissemination.

capable of supporting tissue remodeling and lym-

1. Introduction
The tumor microenvironment (TME) plays an
important role in numerous malignancies and may
even provide a tumor supportive milieu. The TME
contains a wide variety of cytokines and growth
factors and supports crosstalk between neighboring
cells. Among the stromal cells found in the TME,
tumor-attracted and re-educated macrophages
(Mφ), also designated as tumor-associated Mφ
(TAM), have received increasing interest (Manto-
vani and Sica, 2010; Twum et al., 2017). They are
involved in various aspects of tumor progression
like aberrant cytokine secretion, immune check
point modulation, tumor matrix remodeling, angio-
genesis, and resistance to treatment (Mantovani
et al., 2017, 2002; Mantovani and Sica, 2010).
TAMs have been implicated in the pathogenesis of
various lymphoma subtypes such as Hodgkin lym-
phoma (HL), chronic lymphatic leukemias, follicu-
lar lymphoma, and aggressive non- HLs such as
Burkitt lymphoma and DLBCL (Kridel et al.,
2015b, 2015a; Lenz et al., 2008; Pham et al., 2018;
Steidl et al., 2011; Steidl et al., 2010; Verdie et al.,
2018).
Hodgkin lymphoma is a paradigm of a TME-
dominated tumor that is characterized by a very
small portion (< 1%) of malignant cells surrounded
by abundant supportive cells, among which T cells
often dominate (Aldinucci et al., 2010; K€ uppers,
2009; Scott and Steidl, 2014; Steidl et al., 2011).
Additionally, Mφ are an integral part of the TME,
and the number of CD68-positive Mφ predicts the
patient’s outcome (Scott and Steidl, 2014). There-
fore, targeting the interaction between HL cells and
Mφ presents a promising strategy in the treatment
of HL (Locatelli et al., 2018; Ruella et al., 2017).
However, our knowledge of the functions and
interactions of Mφ in HL is still limited (Manto-
vani et al., 2002; Martinez and Gordon, 2014;
Steidl et al., 2010). The present study sheds new
light on the molecular interaction between HL cells
and TAMs and shows that HL-educated TAMs are
phoma dissemination involving the mannose recep-
tor 1 (MRC1/CD206).
2. Materials and methods
2.1. Cell lines, conditioned media, reagents, and
functional assays
The HL cell lines L428, KM-H2, L1236, L540, and
HDLM-2 were provided by V. Diehl (Cologne). The
DLBCL cell lines HBL-1 and OCI-LY3 were
obtained from D. Krappmann (Munich) and the
Deutsche Sammlung f€ ur Mikroorganismen und Zel-
llinien (DSMZ; Braunschweig, Germany), respec-
tively. All cell lines were maintained in Roswell Park
Memorial Institute (RPMI) 1640 supplemented with
2 mg
mL1 glutamine and 10% FCS. At regular
intervals, we used a highly specific Ig PCR to test
for cell identity. In addition, L428, L1236, KM-H2,
OCI-Ly3, and HBL1 cells had been subjected
recently to STR profiling by the DSMZ (Braun-
schweig, Germany).
For the production of lymphoma-conditioned media
(CM), cells were seeded at a density of 5 9 105 cells
per mL and incubated in complete RPMI 1640 med-
ium for 2 days. Cell supernatants were centrifuged at
300 g for 10 min at 4 °C, sterile-filtered, and stored at
4 °C for a maximum of 2 weeks.
2.1.1. Monocyte isolation
Peripheral blood mononuclear cells (PBMCs) of
healthy donors were isolated from fresh buffy coats by
density-gradient centrifugation over Biocoll separating
solution (Biochrom, Berlin, Germany). CD14+ mono-
cytes were obtained from PBMCs by magnetic cell sep-
aration using CD14 microbeads (Miltenyi Biotec,
Bergisch Gladbach, Germany) according to the manu-
facturer’s instructions. Purity of CD14+ cells after
magnetic cell separation was determined by staining
with specific markers and quantification by flow

572 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Arlt et al.
cytometry using a FACSCanto II (BD Biosciences,
Franklin Lakes, NJ, USA).
2.1.2. Macrophage differentiation
Monocyte isolation and macrophage differentiation
were performed as described previously (Menck et al.,
2014). Monocytes were differentiated either in the
presence of RPMI 1640 containing 10% (v/v) FCS,
Pen/Strep, and 2.5 ng
mL1 recombinant M-CSF
(Immunotools, Friesoythe, Germany) or in lymphoma-
CM mixed in equal parts with complete RPMI1640.
Briefly, M-CSF or CM of the tested lymphoma cell
lines was used to incubate primary human monocytes
for 7 days in Teflon-coated cell culture equipment.
After 7 days, cells were counted, harvested, and trans-
ferred into cell culture dishes for additional functional
analysis. Freshly isolated monocytes or Mφ were
stained with cell-specific antibodies or isotype controls.
Expression was quantified by flow cytometry using a
fluorescence-activated cell sorting (FACS) Canto II
and calculated by dividing the mean fluorescence
intensity (MFI) by the MFI of the corresponding iso-
type control. For intracellular CD68 staining, cells
were fixed and permeabilized using the Cytofix/Cytop-
erm Kit (BD BioSciences) following the manufac-
turer’s instructions.
For migration assays, a Boyden chamber with a 5-
µm porous membrane was used (Neuro Probe Inc.,
Gaithersburg, MD, USA). 5 9 104 monocytes were
seeded per well in 50 µL of RPMI 1640 medium and
allowed to migrate for 2 h toward RPMI 1640 supple-
mented with 1% or 10% (v/v) FCS or lymphoma-
CM. The cells that had migrated into the lower cham-
ber were counted.
To analyze endocytosis, Mφ were allowed to adhere
overnight on tissue culture dishes. Cells were washed
twice with PBS and maintained in RPMI 1640 con-
taining 10% (v/v) FCS and pen/strep. 10- and 70-kDa
FITC dextran (at 1 mg
mL1 for 2 h; Sigma Aldrich,
Munich, Germany), respectively, or Gelatin Oregon
GreenTM 488 Conjugate (at 5 µg
mL1 for 30 min;
Thermo Scientific, Waltham, MA, USA) were added
to each plate and incubated at 37 °C or on ice. After
incubation, cells were washed twice with PBS, har-
vested using trypsin/EDTA, and fixed in 2% formalde-
hyde solution in PBS. Uptake was analyzed by means
of a FACS Canto II and calculated by dividing the
MFI of the 37 °C sample by the corresponding MFI
of the sample incubated on ice.
The activity of matrix metalloproteinase (MMP)-9
was determined in cell culture supernatants by gelatin
zymography. First, proteins were separated by SDS/
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Hodgkin lymphoma-educated macrophages and CD206
PAGE. Gel mixes were used to prepare 8% acrylamide
gels containing 1% gelatin and 5% acrylamide stacking
gels. Fifteen microliters of cell culture supernatant has
mixed with an equal volume of loading buffer (62.5 mM
Tris/HCl, pH 6.8, 4% SDS, 25% glycerol, 0.01% bro-
mophenol blue) and loaded onto the gel. Electrophore-
sis was performed under cooling using freezer packs.
Afterward gels were incubated in wash buffer (50 mM
Tris/HCl, pH 7.5, 5 mM CaCl2, 2.5% Triton X-100) for
1 h. Gels were transferred to renaturation buffer (2.5%
Triton X-100) and incubated 1 h. Afterward gels were
covered in development buffer (50 mM Tris base,
150 mM NaCl, 10 mM CaCl2) and incubated overnight
at 37 °C with soft agitation. To visualize the gel degra-
dation by MMP activity, the gels were then stained in
staining buffer (0.5% Coomassie blue, 40% methanol,
10% acetic acid) for 1 h, followed by destaining in
destaining buffer (40% methanol, 10% acetic acid) for
1.5 h. Gels were then fixed for 30 min in fixation buffer
(5% glycerol, 30% methanol), placed between two cel-
lophane membranes and dried overnight. Fixed and
dried gels were scanned for image processing.
Recombinant M-CSF (ImmunoTools) and IL-13
(PeproTech, Hamburg, Germany) were used at 2.5 and
10 ng
mL1, respectively. Inhibitors: the pan-JAK inhi-
bitor pyridone-6 was obtained from Merck (Darmstadt,
Germany), while ruxolitinib, BKM120 (NVP-BKM120),
and ibrutinib ((PCI-32765) were purchased from
Absource Diagnostics GmbH (Munich, Germany).
2.1.3. Fluorescence-labeled antibodies
FITC mouse anti-CD1a (HI149), FITC mouse anti-
CD11b (LT11), FITC mouse anti CD11c (BU15),
FITC mouse anti-CD31 (MEM-05), FITC mouse anti-
CD33 (HIM3-4), FITC mouse anti-CD40 (HI40a),
FITC mouse anti-CD44 (MEM-85), FITC mouse anti-
CD54 (1H4), FITC mouse anti-CD80 (MEM-233),
FITC mouse anti-CD86 (BU63; ImmunoTools), FITC
mouse anti-CD14 (M5E2), FITC mouse anti-HLA-DR
(G46-6), phycoerythrin (PE) mouse anti-CD68 (Y1/
82A; BD Bioscience), allophycocyanin (APC) mouse
anti-CD163 (GHI/61), APC mouse anti-CD206 (15-2),
APC mouse anti-PDL1 (29E.2A3; BioLegend, San
Diego, CA, USA) and isotype controls FITC mouse
IgG1 (PPV-06), FITC mouse IgG2b (PLRV219;
ImmunoTools), FITC mouse IgG2a (G155-178), PE
mouse IgG2b (27-35; BD BioScience), APC mouse
IgG2b (MPC-11; BioLegend) were used for flow
cytometry.
Primary mouse anti-CD30 (Ber-H2) and mouse anti-
CD68 (KP1), anti-Prox1, anti-CD206 (D-1; Santa
Cruz Biotechnology Inc., Dallas, TX, USA), and
573

Hodgkin lymphoma-educated macrophages and CD206
secondary goat anti-mouse horseradish peroxidase
(HRP) polyclonal; Agilent, Santa Clara, CA, USA)
were used for peroxidase and immunofluorescence
staining.
ELISA kits for the detection of secreted M-CSF and
IL-13 in cell culture supernatants were purchased from
R&D Systems (Minneapolis, MN, USA). The sensitiv-
ity was 11.2 and 13.2 pg
mL1, respectively.
2.1.4. Immunohistochemistry on FFPE sections and
immunofluorescence on cryotissue sections
Conventional immunohistochemical staining of CD30,
CD68, and CD206 was carried out according to stan-
dard procedures including endogen peroxidase blocking
(10 min in methanol/H2O2) and antigen retrieval (3 min
at 100 °C in 10 mM citrate buffer, pH 6.0) prior to
incubation with the antibodies. Detection was per-
formed using the ZytoChemPlus (HRP) Polymer Bulk
Kit (Zytomed Systems GmbH, Berlin, Germany). The
counterstaining was performed with Hemalaun after
Meyer (1 : 4; Merck) for 3–5 min and rinsing with
water for 10 min. After dehydration, the sections were
covered with the mounting medium Pertex (Histolab
Products AB; G€ oteborg, Sweden). Immunofluorescence
detection of Prox1 (1 : 500, Reliatech, Wolfenb€ uttel,
Germany), CD30, and CD68 (Dako/Agilent, Hamburg
Germany) was performed on 12-µm cryosections.
Appropriate Alexa Fluor-conjugated secondary anti-
bodies were used (1 : 200; Life Technologies, Eugene,
OR, USA). Immunofluorescence staining of CD163 and
CD206 in tonsil and HL tissue sections was performed
using anti-CD163 antibody (1 : 20, AM10011PU-S rab-
bit, ACRIS/ OriGene Technologies GmbH, Herford,
Germany) or the above-mentioned anti-CD206 anti-
body (1 : 100) in Antibody Diluent (Medac GmbH,
Wedel, Germany), and subsequent staining with corre-
sponding secondary antibody (Donkey anti-mouse
Alexa 555 and Donkey anti-rabbit Alexa 488 [1 : 100,
Invitrogen/Thermo Fisher Scientific GmbH, Dreieich,
Germany)] and 40 ,6-diamidino-2-phenylindole (1 : 5000,
Invitrogen/Thermo Fisher Scientific GmbH). For each
staining step, 100 µL of the respective dilution pro
object slide was used. The anti-CD206 staining of
TMAs was performed with the above-mentioned anti-
CD206 antibody and Histofine anti-Mouse Histofine
anti-mouse and anti-rabbit (Medac GmbH) and stained
with 3,3’-diaminobenzidine (Dako/Agilent Germany).
For tissue microarray (TMA) staining, 200 µL of each
dilution was used.
Slides were scanned by a Hamamatsu Nanozoomer
2.0 RS slide scanner (Hamamatsu Photonics, Ammer-
see, Germany) with 409 magnification for the TMAs
574 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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A. Arlt et al.
and a 209 magnification for the fluorescent-labeled
slides of HL and tonsils. Raw image data were saved
in.ndpi format and handled by the software kit
NDP.VIEW 2 (Hamamatsu Photonics) to save details of
the whole image in.jpeg format. Figures were made
with Adobe Photoshop CS4 Extended (Adobe Systems
Incorporated, San Jose, CA, USA). Chick chorioallan-
toic membrane (CAM) tissue slides were scanned with
a 209 objective (UPlanSApo, NA 0.75) using the vir-
tual slide microscope VS120-L100 (Olympus, Ham-
burg, Germany) equipped with a VC-50 camera.
The experiments and analysis were undertaken with
the understanding and written consent of each subject.
The study methodologies conformed to the standards set
by the Declaration of Helsinki. The study methodologies
were approved by the local ethics committee [Medical
Faculty of the Georg-August-University G€ ottingen (No.
16/5/18An); Medical Faculty of the Christian-Albrechts-
University Kiel, Germany (No. 447/10)].
2.2. Chick chorioallantoic membrane (CAM)
assay
Chicken eggs (Valo BioMedia GmbH, Osterholz-
Scharmbeck, Germany) were bred and regularly turned
for 3 days at 37.8 °C and 80% humidity as described
recently (Klingenberg et al., 2014a; Klingenberg et al.,
2014b).On day 3, a window was sawed into the egg
and sealed with sellotape. On day 10, 2 9 106 L428
cells with or without 1 9 106 CD14+ PBMCs or corre-
sponding 1 9 106 Mφ embedded in 20 µL Matrigel
(BD Biosciences) were inoculated onto the CAM.
After 4 days of incubation, tumors were cut out, fixed,
and embedded in paraffin or Tissue Tek (PolySciences
Inc., Warrington, PA, USA) and cut into 3- or 11-µm-
thick sections, respectively.
In order to access tumor volume and blood content,
specimens were stained with phosphotungstic acid
(PTA) according to the protocol described recently
(Dullin et al., 2017). Then, they were embedded in 1%
agarose gel in 2-mL plastic tubes and scanned using
the in vivo microcomputed tomography (micro-CT)
QuantumFX (Perkin Elmer Health Sciences, Hopkin-
ton, MA, USA) and the following acquisition parame-
ters: 90-kV tube voltage, 200-µA tube current, FOV
20 9 20 mm2, 2-min total acquisition time resulting in
3D datasets with a voxel size of 40 9 40 9 40 µm3.
The software SCRY v6.0 (Kuchel & Sautter GbR,
R€otenbach, Germany) was used for 3D rendering and
volume measurement. For this purpose, the CAM
around the tumor was manually removed using a vir-
tual scalpel and the tumor mass was segmented based
on a brightness threshold.

A. Arlt et al.
2.3. Transcriptomics
The samples were analyzed by RNA-Seq. Read quality
was assessed with fastQC (Andrews (2010): FastQC: a
quality control tool for high-throughput sequence
data. Available online at: https://www.bioinformatic
s.babraham.ac.uk/projects/fastqc/), FastqPuri and
QoRTs (Hartley and Mullikin, 2015; Perez-Rubio
et al., 2018). First, we removed reads perfectly match-
ing the human ribosomal repeating unit (GenBank
accession U13369.1) with FastqPuri. Sequences were
then filtered and trimmed based on quality scores
(bases with quality scores below 28 were removed from
the ends and the remaining read accepted if it had
fewer than 5% base qualities below 28). Then, unde-
termined bases (Ns) were removed from the reads and
the largest N-free sequence was kept as long as it was
at least 25 nt long. Transcript counts were generated
with Kallisto using the Ensembl homo sapiens genome
(release 87). The mean pseudo-alignment rate was
87.42%. In order to mitigate the donor effect, the
combat function of the R-package sva was applied
(Leek J.T. (2018): Available online at: http://biocond
uctor.org/packages/release/bioc/html/sva.html). DESeq2
was used for differential gene expression (GE) analysis
and P-values were adjusted for multiple testing using
the false discovery rate (Hartley and Mullikin, 2015).
KEGG pathway analysis was performed using the R-
package gage (Luo et al., 2009). Variance stabilizing
transformation implemented in the R-package DESeq2
was applied to normalize the data for t-SNE plots. Sub-
sequently, t-SNE dimension reduction was performed
using the R-package Rtsne (Maaten and Hinton, 2008;
Krijthe (2015): Available online at: https://cran.r-projec
t.org/web/packages/Rtsne/citation.html). GE data are
available at NCBI project number PRJNA592308.
2.4. Proteomics
The samples (25 µg per replicate) were subjected to
tryptic digestion using the GASP protocol (Fischer
and Kessler, 2015). Five micrograms of the resulting
peptide mixtures were spiked with 100 fmol of the
retention time standard RePLiCal (Polyquant GmbH,
Bad Abbach, Germany) and analyzed using an Eksi-
gent ekspertTM nanoLC 400 system coupled to a Triple-
TOF 5600+ mass spectrometer (SCIEX, Framingham,
MA, USA).
Five microliters of sample was loaded onto a
YMC-Triart C18 trap column (3 lm particle size,
0.5 cm length; YMC America, Inc., Allentown, PA,
USA) at a flow-rate of 10 µL
min1 for 5 min (iso-
cratic conditions A: 0.1% formic acid, 0.1% ACN).
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Hodgkin lymphoma-educated macrophages and CD206
Peptides were then separated on a reverse-phase col-

umn (YMC-Triart C18, 1.9 µm particle size, 120 A,
1
flow-rate of 5 µL
min , 40 °C) using a 94-min binary
acetonitrile gradient (3–40% B in 87 min, 40–45% B
in 7 min).
The sequential window acquisition of all theoretical
fragment-ion spectra (SWATH) runs was accomplished
using a 50 ms full-MS scan from 400–1000 m/z and 60
subsequent SWATH windows of variable size for
35 ms each (mass range, 230–1500 m/z).
The respective libraries were generated from pooled
samples measured in data-dependent acquisitions mode
(DDA) using the TOP20 method with a full-MS scan
for 250 ms and MS/MS scans for 50 ms each. MS/MS
spectra from the DDA runs were searched against the
respective UniProt database (swissprot-human09-2017)
using ProteinPilot 5.0 and imported in PeakView 2.1
using the SWATH MicroApp 2.0 allowing six peptides
per protein and five transitions per peptide. Raw val-
ues were normalized to total intensity.
The MS proteomics data and search output file have
been deposited to the ProteomeXchange Consortium
(Perez-Riverol et al., 2019) via the PRIDE partner
repository with the dataset identifier PXD01612.
2.5. Combining transcriptome and proteome
data
Filtering for matching proteins and genes in the com-
bined transcriptome and proteome data yielded 1938
gene–protein pairs. Further filtering for genes and pro-
teins that both differed (Padj < 0.01) in expression
between DLBCL and HL stimulated Mφ brought
down the number to 13 gene–protein pairs (Table 1,
top). Analogously, comparing HL and M-CSF-stimu-
lated Mφ revealed 10 gene–protein pairs (Table 1, bot-
tom). In both comparisons, CD206 was the most
differential protein/gene with a log2 fold change (LFC)
of ~ 3.
In addition, differential genes and proteins are listed in
Tables S1-S5: geneExpression_deseq_hl_vs_mcsf.csv,
geneExpression_deseq_hl_vs_dlbcl.csv, proteomics_
limma_hl_vs_mcsf.csv, proteomics_limma_hl_vs_dl-
bcl.csv, proteomics_limma_dlbcl_vs_mcsf.csv.
2.6. Statistical and regression analyses
Results are shown as mean or as mean  SD of the
indicated number of samples. The statistical signifi-
cance of the values was determined using the Student’s
t-test. If applicable, group results were compared by
ANOVA (one-way or two-way analysis of variance)
with subsequent Bonferroni’s post hoc test to correct
575
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Hodgkin lymphoma-educated macrophages and CD206 A. Arlt et al.

Table 1. List of genes that were significantly differentially regulated between L428-educated Mφ and DLBCL- and M-CSF-educated Mφ,
respectively, at both the gene and the protein level.

LFC GE adj.P-value LFC protein adj.P-value Description

L428 vs. DLBCL

ALOX5AP 3.7942 1.17E-49 2.402749983 3.77E-05 Arachidonate 5-lipoxygenase-activating protein
MRC1 3.0765 1.84E-13 2.88006277 1.55E-08 Macrophage MRC1
UPP1 1.7246 2.88E-11 0.835701997 0.000141198 Uridine phosphorylase 1
EPHX1 0.97398 2.95E-08 0.447050392 0.007422156 Epoxide hydrolase 1
HVCN1 0.94723 1.97E-10 0.808504269 0.009561778 Voltage-gated hydrogen channel 1
TTC39B 0.89962 1.56E-06 0.758481005 0.003038771 Tetratricopeptide repeat protein 39B
IL1RN 0.7755 0.0027549 1.145794579 0.008503356 Interleukin-1 receptor antagonist protein
TKT 0.69094 4.81E-15 0.585160474 0.007700075 Transketolase
PTGR1 0.35052 0.0074362 0.562922246 0.009561778 Prostaglandin reductase 1
TCIRG1 0.50824 0.0004946 0.524372957 0.009953695 V-type proton ATPase
MAN2B1 0.53211 9.46E-06 0.547919395 0.006243247 Lysosomal alpha-mannosidase
CPT1A 0.75596 4.83E-08 0.662185065 0.008503356 Carnitine O-palmitoyltransferase 1
SRM 0.93767 9.53E-11 0.437485354 0.009953695 Spermidine synthase
L428 vs. M-CSF
MRC1 3.2116 1.14E-09 3.011783544 4.53E-10 Macrophage MRC1
ALOX5AP 2.0344 8.12E-10 1.597124746 0.004082951 Arachidonate 5-lipoxygenase-activating protein
UPP1 1.6381 9.00E-07 0.928778604 0.000540053 Uridine phosphorylase 1
ALDH2 1.6264 3.57E-18 0.876242857 0.004334703 Aldehyde dehydrogenase 2 family member
STOM 1.3435 0.0009476 1.046572022 0.004082951 Stomatin
TTC39B 1.0793 6.08E-06 0.908818503 0.001644753 Tetratricopeptide repeat domain 39B
EML4 1.0054 2.67E-13 1.229500721 0.004082951 Echinoderm microtubule-associated protein-like 4
IL1RN 1.0028 0.0028429 1.427849353 0.001274962 Interleukin-1 receptor antagonist protein
GCA 0.57588 0.0007919 1.034796929 0.001644753 Grancalcin
FUCA1 0.76156 0.0073074 1.154748957 0.001644753 Alpha-L-fucosidase 1

for multiple comparisons as indicated. Normal distri-
bution and homogeneity of variance were tested using
the Kolmogorov–Smirnov test and the F-test, respec-
tively. The Kruskal–Wallis test with Dunn’s post hoc
test was performed for nonparametric testing. Signifi-
cance levels are indicated as *P < 0.05, **P < 0.01
and ***P < 0.001. All statistical analyses and plots
were done using GRAPHPAD PRISM 6.04 (GraphPad Soft-
ware Inc., La Jolla, USA).
3. Results
3.1. Hodgkin lymphoma cell supernatants attract
monocytes and initiate their differentiation into
Mφ characterized by high mannose receptor 1
expression
To gain insight into the capacity of lymphoma cells to
attract and differentiate monocytes into Mφ, the
migration of monocytes toward conditioned media
(CM) from five HL and two DLBCL cell lines was
first investigated by the Boyden chamber assay and
compared to unconditioned medium containing 1%
and 10% FCS, respectively. Only CM from HL cell
lines, with the exception of HDLM-2, exhibited signifi-
cant chemo-attractive activity (Fig. S1A). However, as
exemplified by KMH2-CM, strong chemo-attractive
activity does not necessarily translate into strong Mφ
differentiation capacity (Fig. S1B). This suggests, in
contrast to previous findings (Locatelli et al., 2018;
Ruella et al., 2017), that attraction and differentiation
are distinct processes regulated by different cytokines
or chemokines.
Next, monocytes from three different donors were
differentiated with L428-CM, OCI-Ly3-CM, HBL1-
CM, and M-CSF, respectively. GE of Mφ was ana-
lyzed by RNA-Seq and compared between Mφ treated
with L428-CM, DLBCL-CM, and M-CSF, respectively
(Fig. 1; Table S1 and S2). L428-educated Mφ are dis-
tinct from DLBCL-CM and M-CSF-differentiated Mφ
(Fig. 1). They showed high expression levels of such
typical macrophage markers as CD68 and CD163.
Their expression levels matched those of CCR1, CSF1,
CSFR1, CTSH, CTSL, CXCL16, or CD53, CD164,
CD276, ITGAX, TGM2, TGFB1, and TGFBI, just to
name a few. Among moderately expressed genes were,
among others, CD209 (DC-SIGN), CXCR4, CXCR5,
IFNAR2, IFGR, IGF1R, IL16, IL6R, IL13RA, STAB1,
S1PR2, SPHK1, and STAT1, whereas ADGRE2,

576 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Arlt et al.
● ●●
100 ●●
● expression, which was significantly higher in L428-edu-
Cell lines
● HBL1
t−SNE 2
0 data can be found in the Tables S1-S5.
L428
MCSF
OCILY3
−100
−100 −50 0 50 100
t−SNE 1
Fig. 1. Factors secreted by HL cells induce distinct changes in
global gene expression by Mφ. Shown is a t-SNE plot to visualize
differences in global gene expression between L428 (HL), OCI-Ly3
(DLBCL), HBL-1 (DLBCL), and M-CSF-induced Mφ.
CCL28, CXCL10, CXCL11, CX3CL1, CXCR2, IDO2,
IL15, IL2RB, PDGFB, and S1PR1 were lowly
expressed. More importantly, the most striking differ-
ences in GE were observed for MRC1 (CD206),
MARCO, CCL3, CCL4, CCL18, CCL20, CXCL3,
CXCL5, CXCL8, CD1B, CD38, CD40, CD74, CD274
(PD-L1), CD301 (CLEC10A), CSFR2, CTSC, FLT1
(VEGFR), ICAM1 (CD54), IL10RA, SOCS3, STAT3,
TNFSF13 (APRIL), and VEGFA. Moreover, HLA-
class II molecules were expressed higher in L428-edu-
cated Mφ than in DLBCL-CM and M-CSF Mφ. GE
of PECAM1 (CD31), CD226, and SPARC, in contrast,
did not differ between L428-educated Mφ and M-CSF
Mφ, but was distinctly lower in DLBCL-derived Mφ.
On the other hand, GE of ADAM9, MMP9, CD52,
CD300C, CD300LB, CD302, CTSB, CTSD or
PDGFRA and TNFRSF11A (RANK) was higher in
DLBCL-CM-treated Mφ than in L428-educated Mφ.
Therefore, it is proposed that L428-educated Mφ have
an M2-like phenotype and differ from DLBCL-CM
and M-CSF-educated Mφ.
Next, a gene set enrichment analysis was performed.
A KEGG pathway comparison between M-CSF and
HL-CM-educated Mφ revealed, for example, that Jak/
STAT signaling and antigen presentation are more
pronounced in L428-CM-treated Mφ (Fig. S2).
To gain deeper insight into the functions of the
L428-educated Mφ, label-free data-independent liquid
chromatography–tandem mass spectrometry was used
to determine protein expression levels in Mφ. Protein
levels were then correlated with the corresponding
Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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Hodgkin lymphoma-educated macrophages and CD206
transcript levels. This analysis confirmed, among
others, the striking difference in (MRC1)/CD206
cated Mφ than in both, M-CSF and DLBCL-CM-
derived Mφ (Table 1). Gene and protein expression
Next, we used flow cytometry to compare the
expression of CD68, CD163, CD206, CD14, (HLA)-
DR, CD1a, CD11b, CD11c, CD40, CD80, CD86,
CD274/PD-L1, CD31, CD33, CD44, and CD54
between M-CSF and L428-educated Mφ and mono-
cytes from 12 different donors (Fig. 2). In concordance
with the omics data, CD206 was the most abundant
molecule expressed on the surface of L428-educated
Mφ. We also observed higher expression of CD68,
CD1a, CD80, PD-L1, CD40, CD3, CD44, and CD54,
further supporting the notion that L428-CM-derived
Mφ represent an M2-like subtype distinct from that of
M-CSF-derived Mφ. Additional flow cytometry data
can be found in Fig. S3. It is evident that CD206 is
expressed more homogeneously on L428-CM than M-
CSF educated Mφ. GE analysis of selected M1 (IL8/
CXCL8, IL1ß, and CXCL9) and M2 (MRC1, CSF-1,
and CCL22) markers lend further support to the flow
cytometry data. GE of Mφ stimulated with M-CSF,
LPS/IFNc, IL4/IL13, IL10, L428-CM and L1236-CM,
respectively, was compared to that of unstimulated
monocytes. The GE profiles concord with the M1-/
M2-profiles and further support the view that HL-edu-
cated Mφ can be defined as M2-like (Fig. S4).
In summary, the stimulation of monocytes with
L428-CM or M-CSF changes the abundance of surface
markers commonly used to monitor Mφ differentia-
tion. This shows that after 7 days, mature Mφ are gen-
erated, which belong to the M2 subtype based on high
CD163 and CD206 expression. Compared to M-CSF-
educated Mφ, L428-CM-differentiated Mφ express
more CD1a, CD80, CD40, and PD-L1, which are
involved in T-cell interactions. The increased expres-
sion of CD11c, CD206, CD33, CD44, and CD54, on
the other hand, points toward enhanced cell–matrix or
cell–cell interactions. The other markers analyzed were
not differentially expressed between L428-CM and M-
CSF-treated Mφ. Importantly, CD206 upregulation in
lymphoma-educated Mφ was also observed for L1236
and L540-CM-derived Mφ when compared with HBL1
and OCI-Ly3-CM (Fig. 3A). This further emphasizes
that the ability of HL cells to differentiate monocytes
into M2-like Mφ, which might play important roles in
T-cell interactions as well as cell–matrix or cell–cell
interactions involving the MRC1 (CD206; Taylor
et al., 2005).
577
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Hodgkin lymphoma-educated macrophages and CD206 A. Arlt et al.

Fig. 2. Surface expression of selected proteins on M-CSF or L428-CM-educated Mφ and monocytes. Flow cytometry analysis of surface
protein expression on monocytes and Mφ differentiated with either 2.5 ng
mL1 M-CSF or L428-CM. MFI was calculated by dividing the
MFI of the specific antibody by isotype MFI (mean  SD, n = 12, paired t-test, two-tailed; *P < 0.05, **P < 0.01 and ***P < 0.001).

Since MRC1/CD206 can bind and take up polysac-

3.2. HL-educated macrophages are characterized
by mannose-dependent endocytosis and
increased collagen ingestion
The strong upregulation of CD206 expression in L428-
CM vs. M-CSF differentiated Mφ suggested that the
former might differ in their endocytic capacity. There-
fore, various endocytosis assays were performed. First
carboxylate-modified fluorescence-labeled latex beads
were used to monitor phagocytic activity (Fig. 3B,C).
We observed no differences in the percentage of cells
that took up latex beads or in the number of beads
taken up, indicating that L428-CM and M-CSF-differ-
entiated Mφ do not differ in the uptake of large
particles.
charides, glycosylated proteins, and collagen (Kato
et al., 2000; Taylor et al., 2005), we tested whether
HL-CM-treated Mφ differed from M-CSF-differenti-
ated Mφ in the uptake of polysaccharides and colla-
gen. We incubated the respective Mφ with 10-kDa
FITC dextran in the presence or absence of mannose
and measured the uptake of FITC dextran by flow
cytometry (Fig. 3D). Mφ differentiated with L428-CM
took up significantly more dextran than M-CSF-
derived Mφ. More importantly, addition of mannose
led to a significant inhibition of the dextran uptake in
L428 CM-educated Mφ, whereas M-CSF-induced Mφ
showed only mannose-unrelated dextran uptake. This
also applied to 70-kDa FITC dextran indicating that

578 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
 18780261, 2020, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12616 by EVIDENCE AID - BELGIUM, Wiley Online Library on [19/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
A. Arlt et al. Hodgkin lymphoma-educated macrophages and CD206

A
B

C
D

E F

Fig. 3. MRC1 gene expression, endocytotic activity, and type I collagen uptake by Mφ. (A) Gene expression of the MRC1 (CD206) in Mφ
differentiated with either 2.5 ng
mL1 M-CSF (ctrl) or lymphoma-derived CM as indicated. (B–D) Mφ were differentiated in Teflon-coated cell
culture bags with either 2.5 ng
mL1 M-CSF or L428-CM for 7 days and transferred to cell culture dishes. After 3 h, nonadherent cells were
washed off and adherent cells were incubated with (B/C) latex beads (5 beads/cell) for 2 h at 37 °C or on ice. Fluorescence was measured
by flow cytometry. The overall percentage of phagocytic cells and their percentages as a function of the number of beads taken up were
calculated by subtracting the respective percentages determined on ice from those at 37 °C (mean  SD; n = 12). (D) Mφ were incubated
with 1 mg
mL1 10-kDa FITC dextran for 2 h at 37 °C and on ice. For blocking, mannose was given 10 min prior to dextran. Fluorescence
was measured by flow cytometry. MFI ratios (MFIR) were calculated by dividing the MFI of 37 °C samples by the MFI of corresponding
samples on ice (mean  SD; n = 12, paired one-way ANOVA with Bonferroni’s post-test). (E) Mφ were incubated with 5 µg
mL1
fluorescence-labeled gelatin OG-488 conjugate for 30 min at 37 °C or on ice. MFIR were calculated dividing the MFI of a 37 °C sample by
the MFI the corresponding sample kept on ice (mean  SD, n = 5, paired t-test, two-tailed; *P < 0.05, **P < 0.01). (F) MMP-9 levels in Mφ
differentiated by M-CSF or L428-CM treatment were analyzed by zymography. The cell-free cell culture supernatants were diluted 1 : 40
before applying to the gelatin gel. Experiments using three different donors are shown.

the higher dextran uptake by HL-CM-educated Mφ is
mediated by CD206 (data not shown). Next, we tested
the uptake of type I collagen by adding fluorescence-
labeled gelatin (gelatin OG-488) to Mφ (Fig. 3E).
Interestingly, as observed for dextran, L428-CM-
educated Mφ also showed a higher capacity of colla-
gen uptake, further supporting the notion that CD206
on HL-associated M2-like Mφ may participate in the
remodeling of the lymphoma microenvironment (Mad-
sen et al., 2013).

Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 579

Hodgkin lymphoma-educated macrophages and CD206
We also tested whether HL-educated Mφ secrete the
matrix metalloproteinases MMP2 and MMP9, which
are involved in extracellular matrix (ECM) degrada-
tion. Zymography, using gelatin-containing gels, failed
to detect any activity of MMP2 in supernatants of
HL- and M-CSF-induced Mφ, whereas both secreted
equally large amounts of MMP9 (Fig. 3F). L428 cells,
in comparison, secreted far less MMP9.
In conclusion, HL-CM-differentiated Mφ differ
from M-CSF-induced Mφ in their ability to take up
CD206-specific targets. Further, MMP9 secretion hints
at an active role of TAMs in remodeling the ECM.
3.3. IL13 is present in HL-CM and upregulates
CD206 in monocytes
A distinguishing feature of HL cells is their ability to
express and secrete IL13 and M-CSF (Lamprecht
et al., 2010; Scott and Steidl, 2014; Skinnider and
Mak, 2002; Tudor et al., 2014). Additionally, Mφ are
known to be polarized toward an M2 phenotype by
IL4 or IL13, which includes upregulation of CD206.
However, whether either or both IL13 and M-CSF
drive CD206 expression remained to be elucidated
(Mantovani et al., 2002). Therefore, we isolated
CD14+ PBMCs and stimulated them with M-CSF, IL-
13, or a combination of both in direct comparison
with L428-CM (Fig. 4A). In parallel, the amount of
IL13 and M-CSF secreted by HL cells used in this
study was determined by ELISA. Both L428 and
L1236 secreted large amounts of IL13 and M-CSF,
whereas DLBCL cells secreted neither (Fig. S5). We
monitored MRC1 GE after 6 and 24 h of stimulation.
There was no detectable expression of MRC1 in
unstimulated and M-CSF-stimulated monocytes after
6 h. However, upon stimulation with either IL13 or
L428-CM, MRC1 was expressed at 6 h, and even more
so after 24 h. Addition of M-CSF did not further
increase IL13-induced MRC1 expression. Compared to
IL13 only, L428-CM-induced expression of CD206/
MRC1 was about tenfold higher after 6 h and
remained twofold higher after 24 h. In addition, the
surface expression of CD206 was analyzed after 24 h
and 7 days (Fig. 4B). In concordance with MRC1 GE
levels, surface expression was increased in IL13 and
L428-CM-treated cells after 24 h but not in untreated
controls or M-CSF-stimulated cells. Again, L428 CM-
treated cells expressed about four times more CD206
than IL13-treated cells. Its expression further increased
after 7 days and was then also detectable on unstimu-
lated and M-CSF-treated cells. This suggests the acti-
vation of an intrinsic program, which needs more time
580 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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A. Arlt et al.
to support MRC1 expression, and that HL-derived
IL13 promotes this process.
Having shown that IL13 was involved in the regula-
tion of MRC1 GE and CD206 protein abundance on
the cell surface of Mφ, we tested the importance of
Jak/STAT signaling. We monitored MRC1 GE in
monocytes after 24 h of stimulation with or without
ruxolitinib, which targets Jak1 and 2, or pyridone 6,
which inhibits all four Janus kinases (Fig. 4C; Thomp-
son et al., 2002; Verstovsek, 2013). Both IL13 and
L428-CM induced MRC1 expression as shown above.
The co-incubation with either ruxolitinib or pyridone 6
completely abrogated MRC1 gene activation by IL13
or L428-CM. This argues for a Jak/STAT-dependent
regulation of MRC1 expression. It also supports the
presence of other Jak/STAT regulating factors in
L428-CM, in addition to IL13, that are responsible for
an even higher upregulation of MRC1 in L428-CM-ed-
ucated Mφ. In parallel, an inhibitor screen was per-
formed to estimate not only the impact of ruxolitinib
or pyridone 6 on MRC1 expression but also to test
their impact on the differentiation of Mφ. We per-
formed Mφ differentiation as described above for
7 days in Teflon-coated cell culture bags either with
M-CSF or L428-CM with and without ruxolitinib or
pyridone 6, in comparison to Ibrutinib and BKM120
(Fig. 4D,E). The strongest effect was observed for
Ibrutinib in L428-CM-educated Mφ. The pan-Jak inhi-
bitor pyridone 6 reduced Mφ differentiation to nearly
50%, whereas ruxolitinib seemed to affect only the
outcome of M-CSF-differentiated Mφ. Both M-CSF
and L428-CM-induced Mφ differentiation was inhib-
ited by the PI3K inhibitor BKM120. Our observation
argues for the involvement of the tyrosine kinase
BTK, PI3K and the Jak/STAT pathway in Mφ differ-
entiation. It also indicates that IL13 and IL13-acti-
vated Jak/STAT signaling, albeit important for MRC1
expression and CD206 upregulation, is only partially
involved in L428-CM-mediated TAM differentiation.
Furthermore, our data imply that M-CSF also acti-
vates the Jak/STAT and PI3K pathways, with differ-
entiation being dependent to some extent on BTK.
These similarities and differences between M-CSF and
HL-educated Mφ need to be further dissected in the
future.
3.4. Lymphoma–Mφ interactions in a preclinical
model
To test the interaction of lymphoma cells with mono-
cytes or Mφ in an in vivo setting, we used the CAM
assay as it recapitulates important aspects of human
 18780261, 2020, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12616 by EVIDENCE AID - BELGIUM, Wiley Online Library on [19/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
A. Arlt et al. Hodgkin lymphoma-educated macrophages and CD206

R
B

C
R
R

Fig. 4. CD206/MRC1 is upregulated by IL13 and L428-CM. (A) CD14+ PBMCs were stimulated with 2.5 ng
mL1 M-CSF, 10 ng
mL1 IL-13,
or both, or with L-428-CM for the indicated time. Gene expression of MRC1 was analyzed by qRT–PCR, relative to GAPDH (mean  SD,
n = 6). (B) Monocytes were seeded in Teflon-coated cell culture bags with 2.5 ng
mL1 M-CSF, 10 ng
mL1 IL-13, or both, or with L-428-
CM mixed with an equal volume of fresh medium. Aliquots of Mφ were taken at the indicated time points and stained with anti-CD206
antibody. MFIRs were calculated as described above (mean  SD, n = 6; paired one-way ANOVA with Bonferroni’s post-test; C) CD14+
PBMCs were preincubated with DMSO or inhibitors for 1 h before 10 ng
mL1 IL-13 or L-428-CM were added. Monocytes were cultured
for six additional hours. Gene expression of MRC1 was analyzed by qRT–PCR, relative to GAPDH and MRC1 expression in L428-CM-treated
monocytes (mean  SD, n = 6). (D/E) Mφ were differentiated in Teflon-coated cell culture bags with either 2.5 ng
mL1 M-CSF (D) or L428
CM (E) for 7 days in the presence of DMSO or indicated inhibitors (1 µM), and cells were counted (mean  SD, n = 4/6; paired one-way
ANOVA with Bonferroni’s post-test; *P < 0.05, and ***P < 0.001).

lymphoma as shown recently (Klingenberg et al., lymphoma cells (L428, L1236) in Matrigel and applied
2014a, 2014b). One million Mφ derived by incubation on the CAM. Four days after application, lymphomas
of monocytes with HL-CM were mixed with 2 9 106 were harvested and characterized macroscopically and

Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 581

Hodgkin lymphoma-educated macrophages and CD206
histologically by defining their volume and investigat-
ing the topography of lymphoma cells and Mφ in rela-
tion to lymphatics and blood vessels (Fig. 5).
The first observation concerned the reduced size of
hemorrhages in lymphomas with Mφ (Figs 5A,B and
S6A). This was confirmed by micro-CT using PTA to
enhance the contrast of blood (Cunningham and
Crane, 1966; Saccomano et al., 2018). This may also
explain in part the smaller size of lymphomas co-inoc-
ulated with Mφ compared to lymphomas without Mφ
(Figs 5A,B and S6B). Moreover, immunostaining with
a-CD30 revealed that HL-derived CAM lymphomas
have a more compact morphology, with lymphoma
cells invading the CAM and occupying the whole area
between the upper chorionic and lower allantoic
epithelium (Figs 5C and S6C). Staining CAM-Mφ-
lymphoma specimens with antibodies against CD30,
CD68, and CD206 revealed a compartmentalization of
the tumor (Figs 5D–F and S6D). CD30-positive HL
cells were located in the upper and lower parts of the
tumor and were almost absent in the center. CD68
and CD206-positive Mφ dominated the upper and cen-
tral parts and were present both in the remaining
Matrigel and intermingled with tumor cells that had
invaded the CAM. Comparable results were observed
when applying CD14+ PBMCs together with L428 cells
on the CAM, further supporting the observation that
HL cells support the differentiation of monocytes to
M2-like Mφ with high CD206 expression (Fig. S6E).
A detailed immunofluorescence analysis of consecu-
tive sections using costaining against CD30 or CD68
with Prox1, a marker for lymphatic endothelial cells,
revealed the absence of lymphatic vessels in L428
CAM lymphomas (Fig. 5G). In CAM-Mφ-lymphomas,
lymphatic vessels were regularly observed at the inva-
sive front (Figs 5H and S7). Dissemination of lym-
phoma cells in lymphatics was regularly visible in
CAM-Mφ-lymphomas (Figs 5I and S7).
3.5. CD206 and primary Hodgkin lymphoma
cases
Immunohistochemical analysis of a tissue microarray
of 16 HL specimens showed that a few cases to feature
a large count of CD206-positive cells in the lymphoma
tissue (Fig. 6A–H). Additional immunofluorescence
analysis revealed that while CD163 and CD206-posi-
tive Mφ are rare in the germinal center of reactive ton-
sils, they are readily detected in HL tissues, including
CD163/CD206 double-positive Mφ (Fig. S8).
Analysis of publicly available patient microarray
data revealed a significant and linear trend for high
CD206 expression in HL patients with stage IV
582 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
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A. Arlt et al.
(Fig. 6I). This supports the view that Mφ with high
CD206 and, thus, probably increased matrix-remodel-
ing capacity are characteristic for advanced stages of
classical HL.
4. Discussion
The lymphoma microenvironment plays an important
role in the pathogenesis of HL. TAMs are a regular
element of this microenvironment. Hence, it is essential
to understand the mechanisms that enable HL cells to
recruit and reprogram monocytes into TAMs (Aldin-
ucci et al., 2010; Steidl et al., 2011). However, despite
the negative impact of increased counts of M2-like Mφ
on outcome and treatment response in HL, there is
still more to be learnt about the mechanisms driving
the development of HL-associated Mφ (Scott and
Steidl, 2014). Our data provide a mechanistic insight
into the processes that are involved in the interaction
of HL cells with Mφ. We present a combined analysis
of transcriptome and proteome data to compare lym-
phoma-educated to M-CSF-differentiated Mφ. We
extend recent data and models, which have proposed
CXCL13, CCL5, and M-CSF as central players in the
interaction of HL cells with Mφ (Carey et al., 2017;
Casagrande et al., 2019; Ford et al., 2015; Locatelli
et al., 2018; Ruella et al., 2017; Scott and Steidl, 2014;
Tudor et al., 2014). We demonstrate that Jak/STAT
signaling and, especially, IL13 are involved in the reg-
ulation of the expression of MRC1 and the abundance
of CD206 on the surface of HL-educated Mφ. Thus,
our study expands the role of IL13 from an autocrine
growth factor to a lymphoma-secreted factor that
mediates ECM remodeling and immunosuppression by
HL-educated Mφ (Skinnider and Mak, 2002), which in
turn facilitates the dissemination of lymphoma cells.
We observed significantly higher counts of Mφ upon
differentiation with HL-CM as compared to DLBCL-
CM or M-CSF. Although M-CSF is expressed by the
majority of HL cell lines, and M-CSF is known to
promote Mφ differentiation into M2-like subtypes, the
observed phenotype and the strongly enhanced CD206
surface expression cannot be attributed to M-CSF in
HL-CM. We observed that IL13 is involved in early
MRC1 mRNA upregulation and enhanced CD206
incorporation into the cell membrane. Enhanced IL13
expression is characteristic of HL, and it has been
shown that IL13 can promote Mφ differentiation
(McKenzie et al., 1993; Skinnider et al., 2001). Condi-
tioned media of L428 and L1236 cells, which featured
the highest capacity to attract monocytes and Mφ,
were also characterized by a high potential to activate
MRC1 GE in Mφ. Both HL cell lines secreted large
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A. Arlt et al. Hodgkin lymphoma-educated macrophages and CD206

Fig. 5. Mφ affect lymphoma growth in the chick chorioallantoic membrane (CAM). (A/B) Tumor volumes are reduced in the presence of Mφ.
L1236 and L428 lymphomas were mixed with Mφ, inoculated on the CAM, and harvested after 4 days of tumor growth. To evaluate the
lymphoma outcome, tumor volumes were defined using micro-CT. Above the graphs are representative macroscopic images (with 7.89
magnification). CAM lymphomas (A) and respective CAM-Mφ-lymphomas (B) are shown. PBMCs from four different donors were used (Mφ
A, B, C, D). Note the absence of bleedings in lymphomas with Mφ (mean  SD; scale bar represents 2 mm). (C) Tissue section of L428
lymphoma stained for CD30. (D) Tissue section of L428 lymphoma with Mφ stained for CD30. (E) Tissue section of L428 lymphoma with
Mφ stained for CD68. (F) Tissue section of L428 lymphoma with Mφ stained for CD206. Co-expression of both CD68 and CD206 on Mφ
shows that the M2-like phenotype is maintained. (C–F) Note some remaining Matrigel (white asterisk) in L428 lymphomas with Mφ. Both,
lymphoma cells and Mφ collectively invade the CAM. However, Mφ seem to consist of two populations, one migrating with the lymphoma
cells and the other remaining at the site of application. See also Fig. S6D,E. (G) Staining of cryosections of CAM lymphoma of L428 cells
and (H) L428 cells with L428-CM-educated Mφ with anti-Prox1 (green) and anti-CD30 (red) to visualize lymphatic vessels and L428 HL cells.
Scale bar: 160 µm. (I) Visualization of HL cells [CD30 (red)] within lymphatic vessels [Prox1 (green), white arrows] to demonstrate their
lymphogenic dissemination. Scale bar: 40 µm. See also Fig. S7.

amounts of M-CSF and IL13. The same was true for
HDLM2 cells (Fig. S5), but in contrast to L428 and
L1236 cells, they failed to induce MRC1 GE in Mφ
(Fig. 3A) for reasons yet to be elucidated. However,
the observation hints at other not yet identified Jak/
STAT activating factors that complement IL13. The
latter binds to an IL4Ra-IL13Ra heterodimer, which
intracellularly can interact with JAK1, JAK2, and
TYK2 (Bhattacharjee et al., 2013). The absence of
MRC1 expression in Mφ after ruxolitinib treatment
indicates that either JAK1 or JAK2 rather than TYK2
induces M2-like GE. Thus, IL13 seems to be not only
involved in the autocrine support of HL cells but also
in rebuilding the lymphoma microenvironment
through Mφ differentiation and upregulation of
CD206. This offers novel opportunities for targeting
the lymphoma microenvironment by Jak/STAT inhibi-
tion (Kim et al., 2014). Whether this approach can be
used in combination or extension of immune check-
point inhibitor therapies needs to be evaluated in
future studies.
Preliminary data show the additional involvement of
BTK- and PI3K-regulated pathways, as respective
inhibitors affect both M-CSF- and L428-CM-mediated
Mφ differentiation. This supports the recent observa-
tion of a preclinical model that used RP6530 to target
the interaction between HL cells and Mφ (Locatelli
et al., 2018). We propose that M-CSF induces a

Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 583
 18780261, 2020, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12616 by EVIDENCE AID - BELGIUM, Wiley Online Library on [19/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Hodgkin lymphoma-educated macrophages and CD206 A. Arlt et al.

A B C D

E F

G H

Fig. 6. Different patterns of CD206-positive cells in classical HL, and gene expression of MRC1 (CD206) in cHL patients of different stages.
(A–D) Four examples of a TMA with a total of 16 specimens demonstrating variable staining patterns for CD206 in classical HL. (E–H)
Details of figures A–D. (A, E) Very few positive cells; (B,F) small number of positive cells; (C,G) moderate number of positive cells; (D,H)
high number of positive cells. Scale bar in E-H = 100 µm. Arrows show Hodgkin and Reed–Sternberg cells, which are negative for CD206
but often in close contact with Mφ. (I) MRC1/CD206 expression data obtained by Steidl et al. were correlated with the stage of cHL
patients. A significant linear trend for high CD206 expression for cHL patients with stage 4 is described (ordinary one-way ANOVA, slope
0.3219; P = 0.0170; Steidl et al., 2010).

network of signaling pathways to activate autocrine
Mφ differentiation factors, whereas IL13 directly pro-
motes an early differentiation program. Although ele-
vated M-CSF serum levels have been reported in HL
patients, and HL cells were found to be M-CSF-posi-
tive by immunohistochemistry, the measured M-CSF
amounts in the lymphoma-CM in vitro and the differ-
ences in Mφ counts and phenotype indicate the
involvement of additional factors during HL-specific
Mφ differentiation (Casagrande et al., 2019; Kowalska
et al., 2012; Zheng et al., 1999). Recently, an autoregu-
latory loop between TAMs and breast cancer cells has
been reported (Cassetta et al., 2019). It might be inter-
esting to test whether described factors such as tumor
necrosis factor alpha, SIGLEC1 and CCL8, which is
self-reinforcing through the production of M-CSF,
also participate in the interaction of HL cells with Mφ.
CD206 is a carbohydrate binding receptor expressed
by Mφ, dendritic cells, and lymphatic endothelial cells.
It belongs to a family of endocytic receptors with con-
served structural elements consisting of an N-terminal
cysteine-rich (CR) domain, a fibronectin type II (FNII)
domain, and several C-type lectin-like domains
(CTLDs; Taylor et al., 2005; Wilting et al., 2009).

584 Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

A. Arlt et al.
Through the CTLDs, CD206 can bind to glycoconju-
gates carrying terminal mannose, N-acetylglucosamine,
and fucose residues, while the FNII and CR domain
bind to collagens and sulfated carbohydrates, respec-
tively. CD206 is a highly effective endocytic receptor
that constantly recycles between the plasma membrane
and the early endosomal compartment. Various exoge-
nous and endogenous ligands have been described for
C-type lectins, implicating several functions in home-
ostasis and inflammation. We observed that HL-CM-
educated Mφ were able to take up collagen, which
concords with previous reports on M2-like Mφ (Mad-
sen et al., 2013). This suggests that these M2-like Mφ
play a specific CD206-mediated role in lymphoma tis-
sue reorganization.
Matrix remodeling is a common process in tumor
progression, and the tumor stroma is characterized by
profound proteolytic degradation (Cox and Erler,
2011; Nakayama et al., 2016; Tataroglu et al., 2007).
The most striking feature of the nodular sclerosis sub-
type of classical HL (NSCHL) is the presence of ECM
deposits. These are collagen-rich fibrotic bands sur-
rounding nodules of inflammatory and neoplastic cells.
Of note, L428 cells are derived from NSCHL. The
observed capacity of L428-CM to induce a specific Mφ
subtype with high CD206 expression and collagen
uptake supports the hypothesis of tissue remodeling by
the interaction of lymphoma cells with stromal cells,
and adds an additional element to the previously
observed mast cell infiltration and fibrosis in HL
(Nakayama et al., 2016). It has to be mentioned that
C-type lectins facilitate the endocytosis of particular
antigen types for cross-presentation, but whether anti-
gen uptake by the C-type lectins will result in immuno-
genic or tolerogenic cytotoxic T-cell priming remains
to be studied (Allen and R€ uckerl, 2017). Taking into
account the capacity of HL-CM to enhance CD40 and
PD-L1 cell surface expression on Mφ, and the recently
described close association of HL cells with PD-L1-
positive Mφ, it is reasonable to assume that HL-edu-
cated Mφ modify T-cell functions at different levels
(Carey et al., 2017).
In summary, we hypothesize that the observed
in vitro and in ovo regulation of CD206 in Mφ might
also occur in vivo and contribute to CD206 abundance
in advanced stages of HL. Our data identify CD206 as
a potential target for the modulation of Mφ activation
in addition to the recently discussed targeting of the
CSF1R (Luo et al., 2006; Martın-Moreno et al., 2015;
Pham et al., 2018; Pyonteck et al., 2013; Ries et al.,
2014; Wang et al., 2018; Zhu et al., 2014). Inhibition
of the Jak/STAT pathway targets not only lymphoma
cells directly, but may also affect lymphoma-educated
Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
18780261, 2020, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12616 by EVIDENCE AID - BELGIUM, Wiley Online Library on [19/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Hodgkin lymphoma-educated macrophages and CD206
Mφ development and/or activity. This supports the
simultaneous targeting of both lymphoma cells and
Mφ with PI3K inhibitors, and the use of Maraviroc
for the blocking of CCR5 in these cells (Casagrande
et al., 2019; Locatelli et al., 2018). In line with this,
targeting the pattern recognition receptor MARCO
can alter Mφ polarization and result in reduced tumor
growth (Georgoudaki et al., 2016). This type of repro-
gramming may now be tested in HL, as HL-educated
Mφ show specific high MARCO expression. Further-
more, imaging of TAMs seems to be a valuable
approach and mannose-coated liposomes or nanobod-
ies are tested for their use in monitoring CD206 TAMs
using PET (Azad and Schlesinger, 2015; Locke et al.,
2012; Movahedi et al., 2012).
5. Conclusion
The present study further corroborates the shown
capacity of HL cells to attract and differentiate mono-
cytes into alternatively activated M2-like Mφ. The lat-
ter are characterized by high CD206 expression, which
indicates that HL-TAMs act as immunosuppressive
agents. Further, CD206 is associated with a high
capacity to bind and take up glycoconjugates with ter-
minal mannose residues as well as type I collagen.
Thus, it is likely that CD206 contributes to the remod-
eling of the tumor microenvironment. Our study high-
lights the role of Jak/STAT signaling and, particularly,
the capacity of IL13 to upregulate CD206 expression
on Mφ, thus expanding its role from an autocrine
growth factor in HL to a lymphoma-secreted factor
capable of modulating the tumor microenvironment.
Additionally, the increased expression of CD40 and
PD-L1 argues for a role of HL-secreted factors in the
reprogramming of monocytes/ Mφ to further support
the capacity of HL cells to suppress, for instance, T-
cell-mediated anti-lymphoma responses. The CAM
assay presents a simple preclinical model to study HL
cell–Mφ interactions and the dissemination of HL
cells. Finally, MRC1/CD206 expression data show a
positive correlation with the stage of HL patients.
Acknowledgements
We thank M. Schaffrinski, B. Manshausen, C. Botz-
von Drathen, and M. F€ ulle for their excellent technical
assistance. We are grateful to the members of the con-
sortia ‘MMML-Demonstrators’ and ‘MMML-Myc-
Sys,’ who contributed to the discussion and prepara-
tion of the manuscript.
This work was supported by grants from the Fed-
eral Ministry of Education and Research
585

Hodgkin lymphoma-educated macrophages and CD206
(Bundesministerium f€ ur Bildung und Forschung,
BMBF) within the networks eBio ‘MMML-MYCSYS’
and eMed ‘MMML-Demonstrators’ (BMBF-FKZ
0316166E, 0316166G, 031A428A, 031A428B,
031A428D; DK, RS, JCE, WG, KD, WK). Further-
more, SR and WK were supported by the Deutsche
Krebshilfe through grant no. 70112502. WK, PJO, and
RS received additional funding from the Wilhelm San-
der-Stiftung, grant no. 2018.037.1. PJO was supported
by Interreg V BY-CZ118. DK was also supported by
‘Stiftung der Georg-August-Universit€ at’ (Kubeschka/
Stricker/Wirth—Stiftung)
Conflict of interest
The authors declare no conflict of interest.
Author contributions
AA and FvB performed most of the experiments. AA,
FL, and FvB conducted in vitro analyses of Mφ and
cell lines, while AA, FvB, RSp, MM, MV, CD, GB,
and JW contributed to specific experiments including
flow cytometry, micro-CT analysis of the chick
chorioallantoic assay, and data interpretation as well
as chick chorioallantoic model analysis. KL, WG, KD,
and PO were involved in proteomic analysis and data
interpretation; SRe and WK performed IHC analysis.
JCE, PP-R, TR, and RSPJ analyzed GE data. VB,
JW, TB, FA, and LT were involved in manuscript
writing and the final approval. AA, FL, TP, RS, JCE,
and DK designed the research, analyzed, and inter-
preted data, RS, PJO, JW, and DK wrote the finally
approved manuscript.
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Supporting information
Additional supporting information may be found
online in the Supporting Information section at the end
of the article.
Fig. S1. Hodgkin lymphoma cells secret factors that
attract and differentiate monocytes into macrophages.
Fig. S2. KEGG pathway comparison between M-CSF
and HL-CM-educated Mφ.
Fig. S3. Flow cytometry analysis of surface expression
of CD206, PD-L1, and CD163 on L428-CM and M-
CSF educated Mφ.
Fig. S4. Examples of gene expression of selected M1-
and M2- Mφ markers.

A. Arlt et al.
Fig. S5. Amounts of IL13 and M-CSF secreted by HL
cells.
Fig. S6. Mφ affect lymphoma growth in the chick
chorioallantoic membrane (CAM), blood vessel
destruction and tissue remodeling.
Fig. S7. Dissemination of lymphoma cells in lymphat-
ics in CAM-Mφ-lymphomas.
Fig. S8. CD163 and CD206 double staining in a repre-
sentative tonsil and HL tissue sections.
Table S1. Differential gene expression of macrophages
comparing HL-CM and M-CSF differentiated macro-
phages.
Molecular Oncology 14 (2020) 571–589 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
18780261, 2020, 3, Downloaded from https://febs.onlinelibrary.wiley.com/doi/10.1002/1878-0261.12616 by EVIDENCE AID - BELGIUM, Wiley Online Library on [19/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Hodgkin lymphoma-educated macrophages and CD206
Table S2. Differential gene expression of macrophages
comparing HL-CM and DLBCL-CM differentiated
macrophages.
Table S3. Differential protein abundance of macro-
phages comparing HL-CM and M-CSF differentiated
macrophages.
Table S4. Differential protein abundance of macro-
phages comparing HL-CM and DLBCL-CM differen-
tiated macrophages.
Table S5. Differential protein abundance of macro-
phages comparing DLBCL-CM and M-CSF differenti-
ated macrophages.
589